Dendritic spines are signaling microcompartments that serve as the primary site of synapse formation in neurons, and that house the machinery underlying memory formation. Actin assembly and myosin 2 contractility play critical roles in the maturation of spines from filopodial precursors. Myosin 18A is a myosin 2-like protein expressed from flies to man that lacks motor activity, is sub-stochiometric to myosin 2, and co-assembles with myosin 2 to make mixed filaments (Billington et al., 2015). Myosin 18A is alternatively spliced to create multiple isoforms that contain unique N- and C-terminal extensions harboring both recognizable and uncharacterized protein: protein interaction domains. These observations suggest that myosin 18A serves to recruit proteins to mixed filaments of myosin 2 and myosin 18A. One such protein is the Rac/Cdc42 guanine nucleotide exchange factor (GEF) Beta-PIX, which is known to promote spine maturation by activating the nucleation promoting factors WAVE and WASp, leading to Arp2/3-dependent branched actin filament assembly (Zhang et al., 2005; Saneyoshi et al., 2008; Spence, 2016). Here we show that myosin 18A is highly expressed in cerebellar Purkinje neurons and concentrates in spines along with myosin 2 and F-actin. Myosin 18As spine targeting is driven by both co-assembly with myosin 2 and an actin binding site present in its N-terminal extension. miRNA-mediated knockdown of myosin 18A results in a significant defect in spine maturation that is manifested as increases in spine length and density, and that is rescued by an RNAi-immune version of myosin 18A. Importantly, Beta-PIX co-localizes with myosin 18A in spines (but not when its myosin 18A binding site is deleted), and its spine localization is lost upon myosin 18A knockdown. Moreover, myosin 18A knockdown results in a significant reduction in the amount of F-actin per spine. These and other data argue that mixed filaments of myosin 2 and myosin 18A present within Purkinje neuron spines form a complex with Beta-PIX that promotes the conversion of filopodia precursors into mature spines by activating Arp2/3-dependent branched actin filament assembly downstream of Beta-PIX's GEF activity. Myosin X (MX) is a highly conserved, vertebrate-specific unconventional myosin whose tail domain contains a PIP3-specfic PH domain, a microtubule-binding MyTH4 domain, and an integrin-binding FERM domain. MX has been linked primarily to the formation and maintenance of filopodia (it is commonly referred to as the filopodial myosin), the transport of integrins in the plasma membrane, and the proper positioning of mitotic and meiotic spindles. Interestingly, neurons express both full length MX (FL-MX) and a headless version (Hdl-MX), and both appear to function in different aspects of radial glia migration (which give rise to most neurons and glia in the neocortex). MX has also been implicated in the transport of the netrin-1 receptor DCC to the tips of neurites, thereby regulating axonal path-finding. In our past efforts to define the function of another unconventional myosin (myosin Va) in Purkinje neurons (PN), the master neuron of the cerebellum, we developed novel tools to study this complex neuron. Interestingly, PNs express much higher levels of MX than other CNS neurons. Moreover, PNs are unique in undergoing filopodia-to-dendritic spine conversion without innervation, perhaps because they express high levels of this filopodial myosin. To begin to address the function of MX in PNs, we expressed GFP-tagged FL-MX in developing PNs. Time lapse imaging showed that MX localizes to the tips of dendritic filopodia, and then moves along these highly motile dendritic filopodia until it localizes to dendritic spines. To extend these observations, we have created a MX conditional knockout (cKO) mouse that targets both FL-MX and Hdl-MX. The whole-body MX KO shows partial embryonic lethality, and mice that survive exhibit a variety of defects including small size, fused digits and white belly spotting. Embryonic phenotypes include exencephaly and gross developmental defects. These data demonstrate that MX is critical for mouse embryogenesis, and that it probably plays a pivotal role in neural tube closure. To access the role of MX specifically within PNs, we are crossing our MX cKO mouse with L7-PCP cre mice, which express cre recombinase specifically in PNs. The mice obtained will be subjected to a variety of tests, from measuring animal coordination, to accessing PN structure and function in situ, in slices, and in culture. Together, these approaches should reveal the critical aspects of MX function in this complex neuron. While mixed primary cerebellar cultures prepared from embryonic tissue have proven valuable for dissecting structure-function relationships in cerebellar Purkinje neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (mESCs) have been successfully differentiated into PNs, although the published methods are very challenging as well. The focus of this study was to simplify the differentiation of mESCs into PNs. Using a recently described neural differentiation media, we generate monolayers of neural progenitor cells from mESCs and differentiate them into PN precursors using specific extrinsic factors. These PN precursors are then differentiated into mature PNs by co-culturing them with granule neuron (GN) precursors also derived from neural progenitors using different extrinsic factors. The morphology of mESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length, and spine density. Furthermore, mESC-derived PNs express Calbindin D28K, IP3R1, IRBIT, PLC4, PSD93, and myosin IIB-B2, all of which are either PN-specific or highly expressed in PNs. Moreover, we show that mESC-derived PNs form synapses with GN-like cells as in primary culture, express proteins driven by the PN-specific promoter Pcp2/L7, and exhibit the defect in spine ER inheritance seen in PNs isolated from dilute-lethal (myosin Va-null) mice when expressing a Pcp2/L7-driven miRNA directed against myosin Va. Finally, we define a novel extracellular matrix formulation that reproducibly yields monolayer cultures conducive for high-resolution imaging. Our improved method for differentiating mESCs into PNs should facilitate the dissection of molecular mechanisms and disease phenotypes in PNs.